I need your help please. I am extracting alguae proteins by alkaine solubilsation- isoelectique precipitation. I obtained a very colored extract? I want to clarify the extract? Do you have any Idea? Could I wash the extract by a solvent such as aceton? But my proteins would be denaturated and thier functional properties would be affected.
Thanks in advance,
The alkaline condition probably oxidized the metabolites of algae in the supernatant. Therefore color must be developed. You can clarify the protein content by dialysis or by using desalting cartridges...Ultrafiltration and sequential washing can also help. AS precipitation can also be an option to isolate the proteome which can keep the proteins in their native state. Other than pI precipitation, other techniques will result in denatured proteins.
The query is a bit confusing. The suggestions for the title question were targeting alkaline solubilization followed by pI precipitation. In these conditions, we assume the proteins remain native and properly folded. Organic solvents such as ethanol alter the active structure. Therefore 2nd part of the question regarding the affected functional properties should be excluded. However, the strategy for the efficient cleaning up the proteome may be discussed. If the colorant substances are not your protein but the small molecules faced with the autoxidation due to the alkaline environment, 8 M Urea or 6 M guanidin solubilization of the protein precipitate followed by one of the dialysis/ultrafiltration/diafiltration/desalting approaches which I indicated previously may be applied to get rid of them. On the other hand, you must be sure that non-enzymatic browning does not take place in alkaline conditions. At high pH, proteins are modified by reducing sugars and carbonyl-bearing molecules known as the Maillard reaction and glycation also alters the functionality. This reaction ends as melanoidin polymers (nitrogenous brown heteropolymers). Metalloproteins may also be affected by the extreme pHs and changing colors may be called a major pitfall as well. Additionally, it is also experienced in ultrafiltration applications, enriched proteins are prone to appear as darkened (browning)...I recommend first dividing the source of the problem and designing the further and/or initial steps later on.
Good luck
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